∗Corresponding author:E-mail,kasajima2008@live.jp /kasajima@iam.u-tokyo.ac.jp ;Fax,+81-3-5841-8466.Plant Cell Physiol. 50(9): 1600–1616 (2009) doi:10.1093/pcp/pcp102, available online at fordjournals © The Author 2009. Published by Oxford University Press on behalf of Japane Society of Plant Physiologists.All rights rerved. For permissions, plea email: journals.permissions@oxfordjournals
T he paper derives a simple way to calculate the linear relationships between all parable groups o f
rate constants for de-excitation of Chl a excitation energy. This is done by comparison of the inver values of chlorophyll fl uorescence intensities and is bad on the matrix model of Kitajima and Butler and on the lake model of energy exchange among PSII centers. Compared with the outputs of earlier, similar calculations, the results prented here add some linear comparisons of the relative sizes of rate
constants without the need f or F 0
′ measurement. This enables us to regenerate the same alternative formula to calculate q L as prented previously, in a dif f erent and simple f orm. The same f ormer equation to calculate F 0
′
value from F m ,F m ′ and F 0 values is also regenerated in our
calculation system in a simple f orm. We also apply relaxation analysis to parate the rate constant for non-photochemical quenching ( k NPQ
) into the rate constant or a f ast-relaxing non-photochemical quenching ( k fast )and the rate constant for slow-relaxing non-photochemical
quenching ( k slow )
. Changes in the sizes of rate constants were measured in A rabidopsis thaliana and in rice.
Keywords:Arabidopsis thaliana
• Chlorophyll fl uorescence parameter • Lake model • Relaxation analysis • Rice •Stern–Volmer approach .
Abbreviations :EET ,excited energy transfer ;∆ F v / F m ,decrea of the parameter F v /F m during treatment ;F ,chlorophyll
fluorescence intensity (in general);F m and F m
′,maximum fl uorescence intensities under dark-adapted or light-adapted
states ;F m ″
,maximum fl uorescence intensity during relaxation analysis ;F v / F m ,a chlorophyll fl
uorescence parameter esti-mating the maximal quantum yield of PSII photochemistry ; F 0and F s
, fl uorescence intensities under dark-adapted or light-adapted states ;F 0 ′
, fl uorescence intensity immediately after turning off actinic light, with all PSII reaction centers
open ;F 0 ″
, fl uorescence intensity during relaxation analysis ; k e and k u ,rate constants of q
E quenching and unknown quenching ;IC ,internal conversion ;IS ,intersystem crossing ; k f , k isc and k d
,
rate constants of chlorophyll fl uorescence, intersystem crossing and basal non-radiative decay ;k fast and
k slow
, r ate constants of fast- or slow-relaxing non-photochemical quenching ;k NP , k fi d and k NPQ , rate constants of sum dissipation, basal dissipation and non-photochemical
quenching ;k pi and k p ,rate constants of photochemistry under dark-adapted or light-adapted states ;k si and k s ,rate constants of the sum de-excitation under dark-adapted or light-adapted states ;LED ,light-emitting diode ;NPQ ,a chlorophyll fl uorescence parameter estimating the size of non-photochemical quenching relative to the size of basal dissipation ;PAM ,pul amplitude modulation ;Φ Fast and
Φ Slow ,chlorophyll fl
uorescence parameters approximating the quantum yields of q E quenching and unknown
quenching ;Φ ISC ,a hypothetical chlorophyll fl
uorescence pa
r ameter estimating the quantum yield of intersystem crossing ;Φ II ,
Φ NPQ and Φ NO , chlorophyll fl uorescence parameters estimating the quantum yields of PSII photochemistry, non-photochemical quenching and basal dissipation ;PPFD ,photosynthetic photon flux density ;q L
and q P ,chlorophyll fl
uorescence parameters estimating the fractions of PSII centers in open states bad on the ‘lake
Estimation of the Relative Sizes of Rate Constants f or Chlorophyll De-excitation Process Through Comparison of Inver Fluorescence Intensities Ichiro Kasajima 1 , 2 , 4 , ∗,Kentaro Takahara 1,Maki Kawai-Yamada 2 , 4and Hirofumi Uchimiya 1 , 3 1 I nstitute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan不可沽名学霸王
2 G raduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570 Japan
3 I wate Biotechnology Rearch Center, 22-174-
4 Narita, Kitakami, Iwate, 024-0003 Japan 4
J apan Science and Technology Agency (JST), Core Rearch for Evolutional Science and Technology (CREST), Saitama, 332-0012 Japan Regular Paper
by guest on March 15, 2012
model’ or ‘puddle model’ of PSII interactions ; q PI ,a chloro-phyll fl uorescence parameter estimating the size of pho-to-chemistry after treatment relative to the size of
photochemistry before treatment ;q S ,a chlorophyll fl uorescence parameter estimating the size of the sum de-excitation under light-adapted states relative to the size of
the sum de-excitation under dark-adapted states ;q Slow ,
a chlorophyll fl uorescence parameter estimating the size of slow-relaxing non-photochemical quenching relative to the
size of basal dissipation ;S ,nsitivity factor ;S fluctuation ,
the hypothetical fl uctuation in the value of nsitivity factor during measurement or treatment.I ntroducti on
M easurement of Chl a fl uorescence parameters by the pul amplitude modulation (PAM) method provides information about the de-excitation fl uxes of Chl excitation energy around PSII, by making non-destructive, simple measure-ments on almost any plant. Fluorescence parameters are cal-culated from veral fl uorescence intensities. The modulation technique measures the increment of total fl uorescence that occurs in respon to a measuring pul. This method enables
measurement of fl uorescence intensities, even under illumi-nated conditions. Fluorescence intensities are measured under different light environments affecting the states of de-excitation fl uxes around PSII ( B aker 2008 ). A typical measurement of Chl fl uorescence with PAM is岩雀出装
illustrated in F ig. 1 . The relative fl uorescence intensity of a dark-adapted plant is designated F 0.F 0 is considered to refl ect both the rate of photochemistry (i.e. the fl ux to pho-tosynthetic electron transport) and the sum of the rates of various basal non-photochemical de-excitations. F m repre-nts the relative fl uorescence intensity of a dark-adapted plant illuminated with a saturating pul. A saturating pul completely reduces components of photosynthetic electron transport for a
moment and stops photosynthetic electron transport, but it does not affect the non-photochemical de-excitations. Thus F m refl
ects the rate of the basal non-photo-chemical de-excitation. The relative fl uorescence intensities under the illumination of a saturating pul are conveniently described as ‘maximum’.
F s reprents the relative fl
uorescence intensity of light-adapted plants which are illuminated with actinic light. The quantum yield of photochemistry is decread and the quantum yield of non-photochemical de-excitation is incread under illumination with actinic light. Under illu-mination, PSII shifts from an ‘open’ state to a partly ‘clod’ state, which means that some of the PSII reaction centers cannot utilize excitation energy under illumination. The increa of non-photochemical de-excitation caud by illu-mination has been usually referred to as ‘non-photochemical
quenching’. The difference between F s and F 0 is caud by
changes in the rates of the de-excitation mechanisms. The
relative maximum fl uorescence intensity of a light-adapted
plant is called F m
′ . The F m ′refl ects the rate of basal non-photochemical de-excitation and the rate of induced non-photochemical de-excitation. F 0 ′
reprents the relative fl uorescence intensity immediately after turning off the actinic light. Supplemental, weak, far-red light to oxidize photosynthetic electron transport fully is provided for a
moment before F 0 ′
is measured. Thus F 0 ′refl ects the rates of dark-adapted photochemistry and of the light-adapted sum
non-photochemical de-excitation. Plea note that F 0 ′
is not measured in
F ig. 1 . Fluorescence measurement in F ig. 1was performed with PAM-101, becau its beautiful trajectory is suitable for illustration. The fl uorescence of veral samples was simultaneously measured with a Clod FluorCam in the other experiments in this paper.
K itajima and Butler (1975) prented a matrix model to provide a linear explanation of fl uorescence intensities by the rate constants of the de-excitation mechanisms. This
matrix model satisfactorily explained the relationship
between t he C hl fl uorescence p arameter F v /F m [ = ( F m – F 0)/F m ]
and the quantum yield of photosynthetic electron transport. F v /F m is now ud as the parameter estimating the maximal quantum yield of PSII photochemistry, which means the quantum yield of PSII photochemistry in the dark. Since
Kitajima and Butler, conditional changes of photochemical and non-photochemical quenching have been discovered
and measured with various Chl fl uorescence parameters. Although there are many parameters to measure various properties of fl uxes around PSII, for example as reviewed by Rohác ˇek (2002), the explanation of the experimental results
is sometimes diffi cult becau many of the parameters lack formal theoretical defi nitions ( B aker 2008 ).
C alculation bad on Kitajima and Butler’s matrix model is a powerful approach for developing Chl fl uorescence parameters which linearly quantify the relative amounts for two groups of rate constants of de-excitation mechanisms. K ramer et al. (2004) adopted this line of attack, generally called the ‘Stern–Volmer approach’, and showed that the relative amount of open PSII is estimated by a new parame-ter q L [=(F m ′ – F s )/(F m ′–F 0 ′)·F 0 ′/F s
] instead of the commonly ud parameter q P .q L is favored rather than q P when the
‘lake model’ fi ts the situation better than the ‘puddle model’ of reciprocal exchange of Chl excitation energy among PSII centers. In the lake model, all PSII centers are hypothesized to be energetically connected with each other to exchange Chl excitation energy. On the other hand, PSII centers are hypothesized to exist as sole independent energy-processing systems in the puddle model. Lake and puddle models are the two opposite extremes. Although an intermediate model between the lake model and the puddle model is consistent with experimental data ( L avergne and Trissle 1995 , L azár 1999 ), K ramer et al. (2004) gave a detailed discussion and concluded that the calculated relative amounts of open PSII are nearly equal between the lake model and the
Detailed calculation of chlorophyll fl uorescence
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intermediate model in terrestrial plants in which the recip-rocal exchange of Chl excitation energy ems to predomi-nate. Thus, calculation bad on the lake model is a good approximation to obtain insight into the sizes of de-excita-tion process bad on simple equations. For non-photo-chemical quenching, they showed that the parameter NPQ
(= F m /F m
′ – 1) estimates the rate constant of induced non-photochemical de-excitation relative to the rate constant of basal non-photochemical de-excitation, by Equation (43) of their paper. In addition to the calculations, they also
showed that the same formula of Φ II
[=(F m ′ – F s )/F m
′],which estimates PSII photochemical quantum yield under illumi-nation, can be derived from both the lake and the puddle models. Parameters for the quantum yield of basal non-photochemical de-ex
citation and induced non-photochemical de-excitation under illumination were also derived { Φ NO = 1/
[NPQ + 1 + q L (F m /F 0 – 1)] and
Φ NPQ = 1 – Φ II – Φ NO }. As appar-ent from the equations, the terms Φ II ,Φ NO and Φ NPQ sum
to 1. This means that the total de-excitation fl uxes of Chl excitation energy can be linearly parated into the three groups. F ollowing the above calculations, H endrickson et al. (2004) propod simple alternative formulae to calculate
Φ NO and Φ NPQ (they call Φ NO as Φ f,D ), such that Φ NO = F s /F m
简单又好看的灯笼and Φ NPQ = F s /F m ′ – F s /F m .Φ NPQ consists of the same formula
as Y N which was propod by L aisk et al. (1997) , thus provid-ing a clear theoretical background for Y N
. The difference in the Φ NO and Φ NPQ formulae between
K ramer et al. (2004) and H endrickson et al. (2004) aris from the difference in
their choices of fl uorescence intensities (whether or not F 0 ′
is ud) and the different ways the formulae are derived. Values derived from the two approaches are esntially the same,
thus calculations of Y N [equal to
Φ NPQ of H endrickson et al. (2004) ] and of Φ NPQ by
K ramer et al. (2004) give similar values ( K ramer et al. 2004 ).
H ere, we propo an improved and easier way to calculate the relative values of rate constants of de-excitation pro-cess. We do this through a comparison of the inver values of the fl uorescence intensities. Although the basic hypothesis of our calculations is esntially the same as tho of previous calculations, the simplicity of our calculation enables an improved understanding of the relationship
F l u o r e s c e n c e i n t e n s i t y (a .u .)
Time (min.)
Fi g.1Illustration of fl uorescence nomenclature and illumination conditions in PAM analysis. Chl fl uorescence of a rotte leaf of A rabidopsis
thaliana was measured. In the fi gure, the x -axis reprents the time-cour of fl uorescence measurement and the y -axis reprents relative fl uorescence intensities. The measuring (modulation) pul was turned on at 0.2 min. At 1.2 min, a saturating pul was supplemented to measure F m . Fluorescence intensity just before this supplementation of a saturating pul corresponds to F 0 . In addition to the measuring pul, actinic light at the photosynthetic photon fl ux density (PPFD) of 300 µmol m –2s –1 was turned on at 2.0 min. During illumination with the actinic light, F m ′ and F s were quentially measured at 3.0, 4.0, 5.0, 6.0 and 7.0 min, then actinic light was turned off at 7.6 min. In the cour of dark relaxation, F m ″ and F 0 ″ were quentially measured at 8.1, 9.1, 10.0, 11.0, 13.1 and 16.0 min, then the measuring pul was turned off at 16.4 min.
扣税计算器I. Kasajima et al.
by guest on March 15, 2012
between fl uorescence intensities and the rate constants of the de-excitation process. Following our method, we are able to calculate the relative amounts between the rate con-stants from only the fl uorescence intensities F 0,F m ,F m
′ and F s ,without F 0 ′ . This elimination of F 0 ′
from calculations is paral-lel to the results of O xborough and Baker (1997) , H endrick-son et al. (2004) and M iyake et al. (2009) . Applications of our calculations to photoinhibition are also described. Finally, possible fl uctuation of the S factor and its effect on the NPQ value are discusd.
R esults T he defi nitions of the de-excitation process of Chl excita-tion energy and its names vary somewhat in the literature and this can be very confusing. To minimize further confu-sion, we here modify tho ud in two recent papers on linear calculations of the relative amounts of the rate con-stants ( H endrickson et al. 2004 , K ramer et al. 2004 ) and we create some new rate constants to make things clearer still. The list of names and the relationships between the rate constants is shown in F ig. 2.
F irst, we create the term ‘sum de-excitation’, which we defi ne as the sum of all the rate constants. This concept is abnt from previous analys but is uful here as it will
help us to gain a better insight into the whole question. We write the rate constant of sum de-excitation as k si and
k s ,where k si reprents the sum de-excitation of dark-adapted plants and k s reprents the sum de-excitation of light-adapted plants (‘i’ means ‘intrinsic’, as in k pi ). Now, k si (or k s )has two components, photochemistry ( k pi or k p
) and sum dissipation ( k NP ).
H ere, the word ‘dissipation’ means ‘energy waste’ as the defi nition of an English word. In terms of de-excitation of Chl excitation energy, all de-excitation process except for photochemistry are energy-wasting process. So, ‘dissipa-tion’ could be equal to ‘non-photochemical de-excitation’. To reprent k NP
, the word ‘sum dissipation’ is ud instead of ‘sum non-photochemical de-excitation’, becau the former phra is shorter. This kind of philosophy was adopted to determine the usage of ven word
s as to six specifi c de-excitation process, and the results are listed in T able 1.The word ‘quenching’ is generally ud for de-excitation pro-cess through intermolecular interactions. Thus ‘quench-ing’ is applicable to photochemistry and non-photochemical
quenching.
k NP is a rate constant, which is the sum of all rate con-stants for dissipation process. Then k NP is further parated
into the sum of basal dissipation ( k fi d ) and non-photochem-ical quenching ( k NPQ
). Basal dissipation is thought to consist of Chl fl uorescence ( k f ), intersystem crossing ( k isc ) and basal non-radiative decay ( k d
) ( K ramer et al. 2004 ). Induced non-photochemical dissipation is hypothesized to consist of three factors, ‘fast’, ‘intermediate’ and ‘slow’ components bad on the relaxation analysis ( Q uick and Stitt 1989 ). Relaxation analysis reprents the measurement of maxi-mum fl uorescence after switching off the actinic light. Fluo-rescence intensities measured in relaxation analysis are
referred to as F m ″
by B aker (2008) . We adopt this usage of the term F m
″ herein (illustrated in F ig. 1 ). Of the three factors of non-photochemical quenching, the fast-relaxing compo-nent is often called q E quenching. q E quenching is depen-dent on the function of PsbS protein ( L i et al. 2000 ). We
write the rate constant of q E quenching as k e
, and the rate constant of the sum of the other unknown non-photochem-ical quenchings as k u . In this paper, we also parate k NPQ into the rate constant of fast-relaxing non-photochemical
quenching ( k fast
) and the rate constant of slow-relaxing non-photochemical quenching ( k slow ).k fast
and k slow reprent experimental approximations of k e and k u values. Of the rate
constants above, all except k fi d ,k f ,k isc and k d are variable
according to the light intensity. In F ig. 2 , we also summarized
the symbols for the quantum yields (
Φ ) of de-excitation process. As described in the Introduction, the quantum yields of de-excitation process are parated into three parts, Φ II ,Φ NO and Φ NPQ .Φ NPQ is further divided into
Φ Fast and Φ Slow herein. T he de-excitation process above are also correlated to the Jablonski diagram of Chl energy states ( T urro 1978 , De
ˇdic et al. 2003, H eldt 2005 , S ugimori 2008 ; F ig. 3 ). Chls at the ground level (S 0 ) are excited to the fi rst singlet state (S 1)
through absorption of red light or to the cond singlet state
(S 2 ) through absorption of blue light. Chls at the cond sin-glet state are unstable and they lo energy in the form of heat by internal conversion (IC) until the fi rst singlet state is reached. The excited Chl can return to the ground state through IC or fl uorescence emittance at the rate of k d and k f .
Energies of fi
rst singlet Chls can also be transferred to photo-chemistry or non-photochemical quenching at the rate of k p
(or k pi ) and k NPQ
. This kind of intermolecular process is called as excited energy transfer (EET). Through intersystem cross-ing (IS), fi
rst singlet Chl can also be converted to the fi rst triplet state (T 1 ), at a relatively low rate ( k isc
). First triplet Chls return to the ground state through phosphorescence
emittance, IS or EET to form singlet oxygen of the
∆ state. In
F ig. 3 , various rotation and vibration energy levels are omit-ted for the sake of simplicity.
B ad on Kitajima and Butler’s matrix model under the lake model of energy exchange among PSII centers, the fol-lowing equation is hypothesized by K ramer et al. (2004) : F = S ûk f / ( k f + k d + k isc + k NPQ + k p )(generally)
(1)Here,F reprents the Chl fl uorescence intensities in gen-eral and S is a constant. Equation (1) is written as a general meaning, and for example the term k NPQ reprents any
Detailed calculation of chlorophyll fl uorescence
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values of k NPQ including zero. This kind of general meaning is
also adopted in Equations (2) and (3). The general equa-tions are ud to show general relationships between fl uores-cence intensities and rate constants. The general equations should be looked at parately from the other specifi c equa-tions, where terms are not shown when their values are zero
and specifi c fl uorescence intensities are given. In all specifi c equations, the same terms have the same values within a t
T able 1 T erminology of the de-excitation process
De-exc i tat i
on process Words k pi k p k f
k isc
k d消防题材电影
k NPQ Quenching O
O
O Dissipation O O O O De-excitation O O O
O
O
O Photochemical O
O
Non-photochemical O O O O Basal O O
O
O
Induced
O
T he applicability of three nouns (quenching, dissipation and de-excitation) and four adjectives (photochemical, non-photochemical, basal and induced) was judged for six de-excitation process. ‘O’ reprents that the word is applicable to the specifi c process.
k : rate constant
Photochemistry (k pi or k p ΦII )
Sum dissipation (k
NP )
q E quenching (k e )
Basal dissipation (k fid ΦNO )
Non-photochemical quenching (k
NPQ ΦNPQ )
Unknown quenching (k u
)
Fluorescence (k f )Intersystem crossing (k isc )Sum de-excitation (k si or k s )
*
***** : de-excitation process with variable rate constants
*Basal non-radiative decay (k d )
(a)
Fast-relaxing non-photochemical quenching (k
fast ΦFast )Slowly-relaxing non-photochemical quenching (k
slow ΦSlow )
**(b)
Φ : quantum yield
F i g. 2 N omenclature and components of de-excitation process. The names of the de-excitation process are shown. For grouped de-excitations, its components are shown below, thus forming a tree-shaped view. The symbols of the rate constants and quantum yields are indicated in parenthes. Two symbols each are shown for rate constants of sum de-excitation and photochemistry. The fi rst symbol indicates the rate constant in the dark-adapted state and the cond symbol indicates the rate constant in the light-adapted state. Variable de-excitation process are indicated by asterisks. Two ways (a) and (b) of dividing non-photochemical quenching are shown; (a) reprents conceptual division and (b) reprents the experimental division performed in this paper.
I. Kasajima et al.
by guest on March 15, 2012
of calculations. Thus specifi c equations are ud to calculate the relationships between fl uorescence intensities and rate constants under each specifi c condition.
E quation (1) is also the fundamental equation in our system. It is important that k p is written as k pi for the dark-adapted state in specifi c equations. As described above,
values of k NPQ and k p change with light intensity. If prented
in simpler and the simplest terms, general Equation (1) is equivalent to the following general equations, respectively: F =S ·k f / ( k fi d + k NPQ + k p )(generally)
win7界面(2) F =S ·k f /k s (generally)(3)
T he difference between general Equations (1) and (2) occurs becau we t the new rate constant k fi d to reprent
the sum of all basal non-photochemical de-excitations. The rate constants of the denominator of the right side of Equa-tion (2) reprent three major groups of de-excitations.
I n general Equation (1), ‘S’ reprents the nsitivity factor, which correlates with the instrument respon (Resp) and light intensity ( I ) to the fl uorescence intensity ( K ramer et al.
2004 ). However, the factor should also contain the propor-tion of incident light that is absorbed by the leaf ( A leaf )and
the fraction of absorbed light that is received by PSII (frac-tion PSII
) ( B aker 2008 ). The proportion of emitted fl uores-cence which is not re-absorbed by Chl (Unabs) should also
戚城遗址be included. Thus, at least fi ve factors are included in S, the nsitivity factor, in our system:
S = I ·A leaf ·fraction PSII ·Unabs ·Resp
(4)
O
ther factors which can be included in S will be also dis-cusd later in this paper. This equation should be applicable to both direct detection and PAM detection of fl uorescence intensity. ‘ I ’ reprents incident light intensity in direct detection and measuring pul intensity in PAM detection. The factor S can fl uctuate, especially under stressful condi-tions ( B aker 2008 ). We refer to such fl
uctuation in S value as ‘S fl uctuation’ in this paper. Becau of the position of the factor S in equations, S fl uctuation caus complex effects on the calculations. Therefore, we will for the moment hypoth-esize that there are no fl uctuations in S during measure-ments as in the ca of the previous calculations. Probable
effects of S fl uctuation on Chl fl uorescence parameters will be discusd later in this paper. F ollowing general Equation (2) and bad on the defi ni-tions of fl
uorescence intensities as described in the cond and the third paragraphs of the Introduction, the specifi
c equations below are derive
d for four reprentativ
e fl uores-cence intensities F 0,F m ,F m ′ and F s :
F 0=S ·k f /(k fi d + k pi )(5) F m =S ·k f /k fi d
(6) F m ′=S ·k f /(k fi d + k NPQ )
(7) F s =S ·k f /(k fi d + k NPQ + k p )
(8)
银行系统
O 2
Photochemistry (k pi or k p ),
Non-photochemical quenching (k NPQ )
Chlorophyll
F i g. 3 J ablonski diagram of Chl energy states. S 0,S 1,S 2 and T 1 correspond to Chl energy levels in the ground state, in the fi rst singlet state, in the cond singlet state and in the fi rst triplet state. Arrows with solid lines indicate excitation process through absorption of red or blue light.
Arrows with broken lines indicate de-excitation process through internal conversion (IC), fl uorescence and phosphorescence emittance,
intersystem crossing (IS) and excited energy transfer (EET). Every corresponding rate constant of de-excitation process from the fi rst singlet state are shown in parenthes.
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